Sahithi Ravuluri1, Rohit Bansal1, Nidhi Chhabra1, Anurag S Rathore2. 1. Department of Chemical Engineering, DBT Center of Excellence for Biopharmaceutical Technology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, 110016, India. 2. Department of Chemical Engineering, DBT Center of Excellence for Biopharmaceutical Technology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, 110016, India. asrathore@biotechcmz.com.
Abstract
PURPOSE: To understand non-enzymatic hydrolytic fragmentation of a monoclonal antibody therapeutic under temperature stressed conditions and investigating possible mechanism for the same. METHODS: The mAb therapeutic was incubated at 50°C in phosphate buffer at pH 6.5 and fragmentation was monitored at different ionic strengths under stressed conditions. The incubated mAb was sampled at regular time intervals by analytical Size Exclusion Chromatography (SEC). RESULTS: It was observed that 57% of the mAb product fragmented over 4 days into two fragment species - Fc-Fab and Fab with molecular weights of 97 KDa and 47 KDa, respectively, as measured by mass spectrometry (MS) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The fragmentation rate was slow initially and then accelerated with time. No change in % aggregate level was observed in this duration, implying that degradation was primarily via fragmentation at high temperature. Kinetics of hydrolytic fragmentation was hypothesized and SEC data was fitted to estimate the kinetic rate constants. While degradation of the monomer into fragment species was non-Arrhenius with a negative activation energy, further degradation of Fab-Fc fragments into Fab or Fc fragments followed Arrhenius Law with an activation energy of 2.1 and 15.38 kcal/mol, respectively. CONCLUSION: High temperature (50°C) causes mAb to cleave at the hinge region to form Fab-Fc and Fab/Fc, as confirmed by dynamic light scattering, SDS-PAGE, SEC, and MS. A kinetic model for hydrolytic fragmentation has been proposed. The results are expected to assist end users in formulation development as well as in monitoring stability of biotherapeutic products.
PURPOSE: To understand non-enzymatic hydrolytic fragmentation of a monoclonal antibody therapeutic under temperature stressed conditions and investigating possible mechanism for the same. METHODS: The mAb therapeutic was incubated at 50°C in phosphate buffer at pH 6.5 and fragmentation was monitored at different ionic strengths under stressed conditions. The incubated mAb was sampled at regular time intervals by analytical Size Exclusion Chromatography (SEC). RESULTS: It was observed that 57% of the mAb product fragmented over 4 days into two fragment species - Fc-Fab and Fab with molecular weights of 97 KDa and 47 KDa, respectively, as measured by mass spectrometry (MS) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The fragmentation rate was slow initially and then accelerated with time. No change in % aggregate level was observed in this duration, implying that degradation was primarily via fragmentation at high temperature. Kinetics of hydrolytic fragmentation was hypothesized and SEC data was fitted to estimate the kinetic rate constants. While degradation of the monomer into fragment species was non-Arrhenius with a negative activation energy, further degradation of Fab-Fc fragments into Fab or Fc fragments followed Arrhenius Law with an activation energy of 2.1 and 15.38 kcal/mol, respectively. CONCLUSION: High temperature (50°C) causes mAb to cleave at the hinge region to form Fab-Fc and Fab/Fc, as confirmed by dynamic light scattering, SDS-PAGE, SEC, and MS. A kinetic model for hydrolytic fragmentation has been proposed. The results are expected to assist end users in formulation development as well as in monitoring stability of biotherapeutic products.
Authors: Zhiqing C Zhu; Yingchen Chen; Michael S Ackerman; Bei Wang; Wei Wu; Bo Li; Linda Obenauer-Kutner; Rulin Zhao; Li Tao; Peter M Ihnat; Jinping Liu; Rajesh B Gandhi; Bo Qiu Journal: J Pharm Biomed Anal Date: 2013-04-28 Impact factor: 3.935